There are 3 major goals for successful optic nerve regeneration and functional recovery: 1) identify more genes that can be manipulated to promote optic nerve regeneration via different mechanisms, 2) identify the optimal combinatory approaches that can promote sufficient regenerating axons from different subsets of retinal ganglion cells (RGCs) to cross the optic chiasm and reach the brain, and 3) precisely guiding regenerating optic nerve axons from different types of RGCs to their original brain targets. The goal 3 is the most difficult one whereas the goal 1 and 2 are the prerequisites to achieve goal 3. The overall goal of this study is to mainly address the first 2 goals. KLF4 and c-Myc, two factors used for the induced pluripotent stem cells (iPSCs) reprogramming, were shown to be important regulators of optic nerve regeneration. The preliminary study showed that overexpression of reprogramming factor Lin28 in mouse RGCs also drastically promoted optic nerve regeneration. A recently completed but unpublished study showed that H3K27 methylation is necessary and sufficient for sensory axon regeneration in vivo by suppressing KLF4. Knocking out the demethylase UTX in RGCs dramatically enhanced optic nerve regeneration. Because H3K27 methylation and associated histone methyltransferase and demethylases have been shown to work together with reprogramming factors during iPSC process by modifying chromatin structure, we hypothesize that mature mouse RGCs can be reprogrammed into a regenerating state via remodeling their epigenetic landscape through reprogramming factors or chromatin modulators. In support, ChIP-seq analysis of H3K27me3 in regenerating neurons identified Magi3, a membrane associated guanylate kinase, as a gene suppressed by H3K27me3. Deleting Magi3 in sensory neurons or RGCs led to marked sensory axon and optic nerve regeneration, respectively. Besides Magi3, many cell reprogramming factors, such as Oct4, FoxA1/2, GATA3/4, PAX6, were identified as top candidate genes regulated by H3K27me3 in regenerating neurons. Therefore, in Aim 1, the study will investigate the roles and mechanisms by which Lin28 and Magi3 regulate optic nerve regeneration. Preliminary study demonstrated that deleting myosin IIA/B in RGCs by itself could promote optic nerve regeneration and abolish backward turning of regenerating axons when combined with Pten deletion. Enhanced RGC neural activity, when combined with mTOR activation, could induce long distance optic nerve regeneration. Thus, in Aim 2, the study will determine if combination of genetic reprogramming with 2 mechanistically different approaches, cytoskeletal modulation or neural activity, can lead to more efficient optic nerve regeneration into the brain. In Aim 3, the study will investigate the potential roles of novel cell reprogramming genes mentioned above in regulation of optic nerve regeneration. The proposed study is based on very strong preliminary data. The results will open a new direction to identify novel genes promoting optic nerve regeneration and build a solid foundation for future functional recovery of vision.